Ž .
Ž After blotting the prawns dry, wet weight to the nearest 0.1 g and total length from
. the tip of the rostrum to the end of the telson were recorded. Females were then
sacrificed and the ovary and MG were removed by dissection and weighed to the nearest Ž
. 0.01 g. Samples of muscle tissue were also collected. The gonado-somatic GSI and
Ž .
midgut gland somatic indices MSI were calculated as the percentage of the weight of the gonad or MG to total body weight, respectively. When tissues of one individual were
insufficient for analysis, tissues were pooled from two to four individuals. All samples were maintained at y20
8C until analysis. 2.1. Lipid analysis
Ž .
The total lipid, lipid class composition and fatty acid methyl esters FAME of the ovary, MG, and muscle tissue were determined using standard analytical procedures.
Ž .
Total lipids were determined according to Folch et al. 1957 , following the modifica- Ž
. tions of Ways and Hanahan 1964 . FAME composition was verified by gas chromatog-
Ž .
raphy according to Coutteau and Sorgeloos 1995 . Lipid classes were analysed using Ž
. high-performance thin-layer chromatography HPTLC as proposed by Olsen and Hen-
Ž .
derson 1989 . 2.2. Data analysis
Differences between the stages of ovarian development were analysed by one-way Ž
. analysis of variance ANOVA and, when pertinent, by Tukey’s honest significant
Ž .
difference HSD test. The homogeneity of the variances of means was checked by the univariate tests of Cochran, Hartley and Barlett. A minimum of three replicates for each
Ž .
tissue was analysed. Results are presented as means standard deviation SD .
3. Results
The biometric data of prawn females are summarised in Table 1. No significant differences were found among wet weight, total length or MSI of females representing
the different stages of maturation. GSI significantly increased during maturation.
Table 1 Weight, total length, GSI and MSI of wild M. rosenbergii females at different stages of ovarian development.
Ž .
Within rows, superscript letters indicate significant differences P - 0.05 Stage of ovarian development
I II
III IV
V Ž .
Prawn weight g 33.412.4
34.310.1 41.412.5
36.711.6 38.810.2
Ž .
Prawn length cm 14.41.1
14.71.4 15.21.9
15.41.7 15.51.4
d cd
c b
a
Ž . GSI
0.480.09 1.510.07
2.790.17 4.390.29
5.920.78 Ž .
MSI 4.030.58
3.910.99 4.280.82
4.110.87 4.420.77
The MG contained the highest levels of lipid among the tissues examined throughout the maturation period, except at stage V when similar levels were found in the ovaries
Ž .
Tables 2 and 3 . Mean lipid content in the MG ranged from 41.5 to 67.0, whereas levels that ranged from 18.2 to 55.8 were found in the ovary. Lipid content of the
Ž .
muscle tissue was generally stable at around 5 Table 4 . Lipid levels in the MG showed a large increase from stages I to III, and tended to decrease between stages III
and V but these changes were not statistically significant due to a wide variance. In the ovary, a significant increase in the content of lipid occurred from stages I to II, but
Ž .
between stages II and IV no significant differences were detected Table 3 . From stages IV to V, a significant increase in ovarian lipid was observed.
The lipid class contents in the MG throughout the maturation period are presented in Ž
. Table 2. Neutral lipids NL were predominant in the MG, comprising from 73.4
Ž .
Ž .
stage I to 79.4 stage III of all lipids. The NL:PL ratio in the MG ranged from 2.8 to 3.9. Total NL increased significantly from stages I to III, but decreased significantly
from stage III to stage V. The overall increase in NL was due mainly to a significant Ž
. increase in triacylglycerols TG , which presented variations similar to NL. At stage V,
TG represented 53.8 of all NL in the MG. Significant differences in sterol and steryl Ž
. ester SE content were also detected in the MG. SE levels were stable between stages I
and II, and followed by ups and downs between stages II to V. Sterols were also stable
Table 2 Ž
. Ž
. Variations in total lipids dry weight and lipid class content of dry weight in the MG of wild M.
Ž .
rosenbergii females at different stages of ovarian development. Each value is the mean SD of four Ž
. separate prawn samples analysed individually in stage III, ns 3 , except for stages I and II where tissues of
Ž .
three prawns were pooled. Within each row, superscript letters indicate significant differences P - 0.05 Stage of ovarian development
I II
III IV
V
c b
a a
a
Total lipids 41.52.2
53.02.3 67.02.9
58.75.2 51.74.1
c b
a ab
b
Total NL 30.50.5
41.31.8 53.21.3
45.14.9 39.44.9
b b
ab ab
a
Sterols 2.10.5
2.20.3 3.30.3
3.21.1 4.31.9
ab ab
ab a
b
Free fatty acids 8.91.8
11.21.5 9.81.1
12.80.7 6.74.0
c bc
a bc
ab
Triacylglycerol 12.63.8
16.21.8 26.92.0
16.96.3 21.23.0
b b
a b
a
Steryl esters 1.20.3
1.70.4 3.20.2
1.70.7 3.30.8
Monoacylglycerolq pigments 2.51.3
4.00.5 3.70.9
4.10.6 1.81.8
bc ab
ab a
c
Diacylglycerol 3.20.3
5.90.7 6.31.7
7.00.9 2.12.8
Total PL 11.00.5
11.71.8 13.81.3
13.64.9 12.34.9
Phosphatidylcholine 4.70.3
4.80.9 6.00.4
6.21.8 6.62.7
Phosphatidylserine 0.60.2
0.70.5 0.50.1
0.40.3 0.20.3
Phosphatidylinositol 1.10.2
1.10.6 1.70.2
1.40.8 0.90.4
Phosphatidylethanolamine 3.00.1
3.20.4 4.40.5
4.41.3 4.01.8
Lyso-phosphatidylcholine 0.90.9
1.00.7 00
0.20.1 0.10.1
ab ab
a a
b
Phosphatidic acid 0.60.2
0.70.2 1.10.2
1.00.6 0.20.0
Sphyngomyelin 0.10.1
0.20.1 0.10
0.10.1 0.10.2
Glycolipids 00
0.10.2 00
00 0.10.1
NL:PL ratio 2.8
3.5 3.9
3.3 3.2
Table 3 Ž
. Ž
. Variations in total lipids dry weight and lipid class content of dry weight in the ovary of wild M.
Ž .
rosenbergii females at different stages of maturation. Each value is the mean SD of three separate prawn samples analysed individually, except for stages I, II and III, where tissues of four, three and two prawns were
Ž .
pooled, respectively. Within each row, superscript letters indicate significant differences P - 0.05 Stage of ovarian development
I II
III IV
V
c b
b b
a
Total lipids 18.21.7
34.82.7 39.53.3
39.32.8 55.82.3
d c
bc b
a
Total NL 10.70.3
21.00.5 24.22.2
25.31.4 33.01.2
d c
b b
a
Sterols 2.00.2
3.10.3 4.00.3
4.20.1 6.30
b a
b b
c
Free fatty acids 2.00.6
3.60.3 2.20.3
1.80.4 0.70.2
d c
b b
a
Triacylglycerol 4.60.8
10.60.5 14.32.2
15.70.8 21.41.5
b a
a a
a
Steryl esters 0.80.3
1.80.1 1.70.3
1.60.1 2.10.1
Monoacylglycerolq pigments 0.80
0.70.1 0.60
0.50 0.60.4
c b
b b
a
Diacylglycerol 0.50
1.20.1 1.40.2
1.30 1.80
c b
b b
a
Total PL 7.50.3
13.80.5 15.32.2
14.01.4 22.81.2
c b
b b
a
Phosphatidylcholine 3.20.6
6.20.6 7.91.2
7.71.0 10.90.9
a a
ab b
ab
Phosphatidylserine 0.60.2
0.70.1 0.50.3
0.10 0.50.1
b a
a b
a
Phosphatidylinositol 0.60.1
1.30.1 1.20.3
0.50.2 1.40.1
d c
bc b
a
Phosphatidylethanolamine 2.00.3
4.00.4 5.00.5
5.10.3 9.00.3
Lyso-phosphatidylcholine 0.10.1
00 00
00 00
ab a
b b
ab
Phosphatidic acid 0.50.1
0.70.2 0.30.1
0.20.1 0.30.1
Sphyngomyelin 0.50.1
0.70.1 0.40.2
0.30.3 0.40.1
Glycolipids 0.10.1
0.10.1 00
00 0.2 0.2
NL:PL ratio 1.4
1.5 1.6
1.8 1.4
Table 4 Ž
. Ž
. Variations in total lipids dry weight and lipid class content of dry weight in the muscle of wild M.
Ž .
rosenbergii females at different ovarian development stages ns 3 . Within each given row, superscript letters Ž
. indicate significant differences P - 0.05
Stage of ovarian development I
II III
IV V
Total lipids 5.20.3
4.00.2 4.60.5
4.80.2 6.40.9
Total NL 2.30.1
1.60.1 1.90.3
2.20 2.50.6
Sterols 1.00
0.70.1 0.80.1
0.90.1 0.90.2
Free fatty acids 0.50
0.30.1 0.30.1
0.30 0.60.3
Triacylglycerol 0.30.1
0.30.1 0.30
0.30 0.30.3
Steryl esters 0.30.1
0.30.1 0.40
0.40 0.40.3
Monoacylglycerolq pigments 0.10
0.10 0.10
0.10 0.20.2
Diacylglycerol 0.10
0.10 0.10
0.10 0.10.1
Total PL 2.90.1
2.40.1 2.70.3
2.60 3.20.6
Phosphatidylcholine 1.10
1.00.1 1.10.2
1.00 1.20.3
Phosphatidylserine 0.40.1
0.30.1 0.30
0.40 0.50.1
Phosphatidylinositol 0.20.1
0.20 0.20
0.20 0.30
Phosphatidylethanolamine 0.90
0.70 0.80
0.90 0.80.3
Lyso-phosphatidylcholine 00
00 00
00 00
Phosphatidic acid 0.20
0.10 0.10
0.10 0.20.1
Sphyngomyelin 0.20
0.20 0.20
0.20 0.20.1
Glycolipids 00
00 00
00 00
NL:PL ratio 0.8
0.7 0.7
0.8 0.8
Ž .
between stages I and II, but increased from stage II to stage V. Free fatty acids FFA and diacylglycerols followed similar up and down trends during the maturation period,
Ž .
but the lowest levels were found at stage V. Total polar lipid PL in the MG did not significantly vary throughout the maturation period. Phosphatidic acid was the only PL
class to present significant variations, with stable levels from stages III to IV, but Ž
. decreasing significantly towards stage V. Phosphatidylcholine PC , phosphatidylserine
Ž .
Ž . Ž
. PS , phosphatidylinositol PI , phosphatidylethanolamine PE , lyso-phosphatidylcho-
Ž .
line LPC , sphyngomyelin and glycolipids showed no significant variations. Table 3 presents the changes in lipid class levels in the ovary during maturation.
Total NL was more abundant in the ovary than PL. At stage I, NL:PL ratio was 1.4 and increased to 1.8 as GSI increased up to stage IV. Total NL levels doubled between
stages I and II and continued to present significant increases throughout maturation. Ž
. Ž
. Most of the NL was TG, which ranged from 43 stage I to 64.8 stage V . Sterols
were the second most abundant lipid class among NL in the ovary, increasing from 2.0 to 6.3 between stages I and V, respectively. SE levels increased significantly from
stage I to stage II and remained stable thereafter. Levels of FFA fluctuated throughout maturation; the lowest level was found at stage V. Diacylglycerol levels increased
significantly from stage I to II, were stable from stage II through IV, and reached a peak value at stage V. Total PL levels in the ovary at stages V were significantly greater than
all other stages. PC and PE were quantitatively the main components of the PL fraction,
Ž .
Ž .
collectively representing from 69.3 stage I to 91.4 stage IV of all PL in the ovary. Levels of both PC and PE were significantly higher at stage V of ovarian
maturation. The level of PI also increased significantly during maturation, but was comparatively lower than either PC or PE. Other PL classes presented either negligible
Ž .
Ž up and down trends PS and phosphatidic acid or no significant variations LPC,
. sphyngomyelin and glycolipids throughout the maturation period.
The lipid class composition of the muscle tissue is presented in Table 4. Unlike the MG and ovary, PL were more abundant than NL with the NL:PL ratio ranging between
0.7 and 0.8. No significant variation was observed for any of the lipid classes throughout maturation. Neither LPC nor glycolipids were detected in the muscles at any stage of
ovarian development.
The fatty acid composition of the MG, ovary and muscle tissue throughout the maturation period is presented in Tables 5, 6 and 7, respectively. The predominant fatty
Ž .
Ž .
Ž .
Ž .
acids in the MG were myristic 14:0 , palmitic 16:0 , stearic 18:0 , oleic 18:1n y 9 Ž
. Ž
. Ž
and linoleic 18:2 n y 6 acids, whereas linolenic 18:3n y 3 , arachidonic 20:4 n y 6; .
Ž .
Ž .
ARA , eicosapentaenoic 20:5n y 3; EPA and docosahexaenoic 22:6 n y 3; DHA acids were present at intermediate levels. Saturated and mono-unsaturated fatty acids in
the MG increased significantly as ovarian development proceeded. In contrast, n y 3 Ž
. highly unsaturated fatty acids HUFA , particularly EPA, decreased. No significant
Ž .
variation in the relative amount of n y 6 poly-unsaturated fatty acids PUFA was detected. Ovarian lipids were composed mainly of 16:0, 18:0, 18:1n y 9 and 18:2 n y 6.
As in the MG, level of saturates and mono-unsaturates increased significantly in the ovary as gonadal development progressed. From stage I to V, the contents of saturates
and mono-unsaturates in the ovary rose by 156.9 and 189.0, respectively. Among
Ž .
poly-unsaturated fatty acids PUFA , the levels of 18:2 n y 6 and 18:3n y 3 significantly
Table 5 Ž
y1
. Principal fatty acid content mg g
dry weight of the MG of wild M. rosenbergii females at different Ž
. ovarian development stages ns 4, except stage III, where ns 3 . Different superscripts within rows represent
Ž .
significant differences P - 0.05 Fatty acid
Stage of ovarian development I
II III
IV V
b b
ab ab
a
14:0 30.88.0
40.325.6 53.65.0
62.533.4 88.028.6
c c
ab bc
a
16:0 72.821.5
90.69.2 120.823.0
99.09.0 127.49.3
18:0 27.912.8
28.63.0 41.410.5
35.74.8 37.87.5
18:1ny9 65.015.9
102.712.0 102.932.1
84.148.1 93.739.4
18:2 ny6 39.715.9
73.532.3 55.716.2
50.458.3 53.211.4
a ab
ab b
b
18:3ny3 6.80.8
5.21.4 4.80.2
4.11.7 3.91.5
20:4 ny6 6.91.2
4.92.2 3.80.4
4.41.4 4.84.3
a ab
b b
b
20:5ny3 6.91.2
4.92.2 3.80.4
4.01.4 2.60.9
22:6 ny3 5.01.1
3.81.8 3.21.5
4.72.8 2.50.7
c bc
ab ab
a
Saturates 143.544.9
177.335.5 234.632.4
213.222.3 268.029.6
b ab
ab ab
a
Mono-unsaturated 86.523.3
127.311.2 140.131.6
99.844.7 144.843.3
ny6 PUFA 49.015.6
80.231.9 63.115.4
55.458.1 58.212.0
a ab
ab ab
b
ny3 HUFA 12.92.3
9.54.6 8.32.0
9.34.4 6.01.8
ny6r ny3 ratio 3.80
8.44 7.60
5.96 9.70
increased from stage I to III, while ARA levels decreased significantly during the same period. A significant increase in the levels of DHA and total n y 3 HUFA was observed
between stages I and II. Muscle tissue contained levels of fatty acids that were lower
Table 6 Ž
y1
. Principal fatty acid content mg g
dry weight of the ovary of wild M. rosenbergii females at different Ž
. ovarian development stages
ns 3 . Different superscripts within rows represent significant differences Ž
. P - 0.05
Fatty acid Stage of ovarian development
I II
III IV
V
c bc
abc a
ab
14:0 3.50.7
5.33.0 7.32.1
16.28.4 12.76.3
d c
b ab
a
16:0 23.67.0
36.710.8 53.33.6
64.11.4 66.58.6
c bc
ab a
a
18:0 9.12.4
13.34.3 17.31.7
19.51.0 18.62.1
c b
ab a
a
18:1ny9 25.57.0
46.47.8 59.711.8
68.04.6 68.89.5
b ab
a a
a
18:2 ny6 17.42.5
41.56.1 50.126.2
47.715.6 43.06.8
b ab
a a
a
18:3ny3 1.20.3
2.61.0 4.12.2
3.81.1 3.71.2
a ab
b b
b
20:4 ny6 12.83.7
9.61.1 7.30.6
6.61.6 6.41.1
20:5ny3 7.92.1
11.23.3 9.02.1
9.03.2 9.03.0
b a
a a
a
22:6 ny3 3.91.3
7.92.2 6.53.3
7.73.1 6.62.5
c c
b a
a
Saturates 39.911.2
59.620.4 82.55.9
104.68.2 102.59.1
c b
ab a
a
Mono-unsaturated 32.69.6
56.511.3 76.515.8
89.812.8 94.210.7
ny6 PUFA 31.45.9
52.47.8 59.325.0
56.314.5 52.07.7
b a
a a
a
ny3 HUFA 12.33.3
20.15.1 17.25.0
18.15.7 17.35.1
ny6r ny3 ratio 2.55
2.61 3.45
3.11 3.01
Table 7 Ž
y1
. Principal fatty acid content mg g
dry weight of the muscle tissue of wild M. rosenbergii females at Ž
. different ovarian development stages ns 3 . Different superscripts within rows represent significant differ-
Ž .
ences P - 0.05 Fatty acid
Stage of ovarian development I
II III
IV V
14:0 0.60.3
0.50.3 0.90.3
0.60.2 0.50.3
16:0 5.61.2
5.91.3 6.00.4
4.60.6 4.90.5
18:0 3.50.9
3.20.7 3.20.2
3.10.3 3.00.5
18:1ny9 5.01.1
5.40.8 6.00.7
5.40.7 5.50.9
18:2 ny6 2.81.0
3.90.5 2.90.5
3.10.5 2.80.5
18:3ny3 0.30.1
0.30.2 0.30.2
0.10.1 0.40.2
20:4 ny6 3.50.2
3.20.6 2.90.3
2.70.5 2.80.3
20:5ny3 3.00.1
2.70.4 2.40.1
2.90.3 2.80.2
22:6 ny3 1.40.2
1.10.3 1.10.3
1.20.3 1.10.3
Ý Saturates 10.82.5
10.52.4 11.00.9
9.30.9 9.41.3
Ý Mono-unsaturated 6.61.6
7.11.3 8.10.6
6.70.9 7.51.4
ny6 PUFA 7.01.5
7.61.2 6.20.8
6.00.9 6.00.5
ny3 HUFA 4.60.2
4.10.2 3.70.4
4.20.6 4.00.4
ny6r ny3 ratio 1.52
1.85 1.68
1.43 1.50
than those of the other tissues examined. No significant variation in the levels of any fatty acid was detected.
4. Discussion
